Cell activity assay apparatus and methods for making and using same

ABSTRACT

A cell activity assay apparatus (“CAAA”) and method using electromagnetic radiation detection beams to measure the cell activity, e.g., chemotactic response, includes an opaque membrane having a plurality of off-axis pores which prevent experimentally significant amounts of the electromagnetic radiation from traversing the membrane. The membrane is fabricated from opaque films that have been either ablated with substantially off axis excimer laser electromagnetic radiation to form pores, or bombarded by substantially off-axis charged particles and then etched to form pores.

BACKGROUND

[0001] 1. Field of the Invention

[0002] The present invention relates to methods and apparatus for cellactivity assay (CAA) investigation of chemotaxis, migration, invasion,angiogenesis, growth, proliferation, differentiation, or interaction ofcells in response to various chemical environments.

[0003] 2. Background of the Invention

[0004] Chemotaxis is the directional movement (migration) of biologicalcells or organisms in response to concentration gradients of chemicals.Invasion is the movement (migration) of cells into or through a barrier.Tumor invasion is such action initiated by cancer cells into or throughbiological tissue in vivo, or, into or through extra cellular matrixproteins, e.g., collagen or matrigel, into or through barriers made ofother substances, in vitro. Angiogenesis is the migration and formationof capillary blood vessels by endothelial cells. Growth is the increasein the size, form, or complexity of cells. Proliferation is growth ofcells by cell division. Differentiation is the process by which cellschange from a less specialized to a more specialized state usuallyassociated with different functional roles and the expression of new anddifferent traits. Interaction of cells is the alteration of cellbehavior such as movement, invasion, angiogenesis, growth,proliferation, or differentiation in response to the presence and actionof nearby cells of the same or different type. These activities andsimilar activities are referred to herein collectively as “cellactivity,” and the apparatus employed to do the assays is referred toherein as “cell activity assay apparatus.”

[0005] One kind of single-site conventional cell activity assayapparatus referred to variously in the literature as “chemotaxischambers,” “Boyden chambers,” “Boyden chemotaxis chambers,” “blind wellchambers,” or “microchemotaxis chambers,” comprises two compartmentsseparated by a membrane, with one or both of the compartments open toair. Multi-site apparatus are referred to as “multi-well chemotaxischambers,” or “multi-well Boyden chambers,” and have the same basic sitestructure but have multiple sites. (See U.S. Pat. Nos. 5,210,021 and5,302,515) Assays employing this kind of apparatus pipette cellssuspended in media into the upper compartments, and pipette chemotacticfactors and controls into the bottom compartments. The chemotacticfactors can be used in various dilutions to get a dose-response curve.The controls are generally of three kinds: (a) negative, when the samemedia that is used to suspend the cells is also used below the membrane,(b) chemokinetic, when a chemotactic factor is placed at the sameconcentration in the media with the cells and in the well on theopposite side of the membrane, and (c) positive, when a knownchemoattractant is placed in the bottom wells. Chemokinetic controlsallow the user to distinguish heightened random activity of the cells,due to contact with the chemotactic factor, from directional response ina concentration gradient of that chemotactic factor.

[0006] Cell activity assay apparatus can also be used to measure theresponse of cells of different origins—e.g., immune cells obtained frompatients suffering from diseases—to a chemotactic factor of knownchemotactic activity. In this case the cells in question areinterrogated by both a negative control and a known chemotactic factorto see if the differential response is depressed or normal.

[0007] Chemotactic activity is measured by establishing a stableconcentration gradient in the cell activity assay apparatus; incubatingit for a predetermined time; and then counting the cells that havemigrated through the membrane (or into the membrane). A comparison isthen made between the activity of the cells in a concentration gradientof the chemotactic factor being tested, and the activity of the cells inthe absence of the concentration gradient.

[0008] In one type of cell activity assay apparatus and method, thechemotactic response is measured by physically counting the number ofcells on the membrane surface closest to the chamber containing thechemical agent. An example of this type of cell activity assay apparatusis described in U.S. Pat. No. 5,210,021 (Goodwin, Jr.), which is herebyincorporated by reference. One prior art method of obtainingquantitative data is to remove the membrane from the cell activity assayapparatus, remove the cells from the membrane surface closest to thechamber containing the original cell suspension, fix and stain theremaining cells, and then observe and count the stained cells under amicroscope. Because of the time and expense associated with examiningthe entire membrane, only representative areas of the membrane arecounted, rendering results less accurate than would otherwise be thecase if the entire membrane were examined and counted.

[0009] Cell activity assays using a disposable ninety-six wellmicroplate format, for example the ChemoTx™ System (available from NeuroProbe, Inc., Gaithersburg, Md.), is amenable to different methods ofquantification of results. The manual staining and counting methoddescribed above can be used, but is not recommended due to the timeinvolved. A preferred method is to centrifuge the microplate with filterattached, such that, the cells that have migrated through the filter aredeposited onto the bottom of the lower wells. The cells are then stainedwith MTT, MTS (available from Promega, Madison, Wis.), or similar dye,and then read in a standard automated laboratory densitometric reader(sometimes referred to as an Elisa plate reader).

[0010] Another method of obtaining quantitative data with this apparatusis to dye the cells with a fluorescent material, e.g., Calcein AM(available from Molecular Probes, Eugene, Oreg.); centrifuge themigrated cells into the microplate, and count cells with an automaticfluorescence plate reader (e.g., Cytofluor available from PE Biosystems,Foster City, Calif., Victor² available from EG&G Wallac, Gaithersburg,Md., or fmax available from Molecular Devices, Sunnyvale, Calif.). Theautomatic plate reader excites the fluorescent dye in the migrated cellswith one wavelength of light and reads the light emitted at a secondwavelength. Alternatively, the cells that have not migrated are removedfrom the top of each site, and the plate with framed membrane attachedis read in the automatic fluorescent plate reader without spinning thecells into the plate, thereby counting the cells that have fallen offthe filter into the lower well as well as those on the bottom of themembrane and in the pores of the membrane.

[0011] In another type of chemotaxis apparatus, illustrated by U.S. Pat.No. 5,601,997 (Tchao), the chemotactic response is also measured bylabeling the cells with a fluorescent dye, as above. However, in Tchaothe membrane is made of film opaque to the excitation and emissionwavelengths of the fluorescent dye so that the cells on one side of themembrane can be counted without removing the cells from the oppositeside. Tchao's method is an example of a kinetic assay. In such assays,the side of the membrane toward which the cells are migrating isilluminated with the excitation wavelength of the dye, and the cells onthat side are periodically counted by measuring the intensity of lightemitted in the emission wavelength. This gives the researcher data onthe rate at which cells are moving through the membrane. The membranemust be opaque because the researcher cannot remove the cells from theside from which they originated without ending the assay, which makes akinetic study impossible.

DEFINITIONS & ABBREVIATIONS Abbreviations

[0012] 1. “Electromagnetic radiation” is herein abbreviated to “ER.”

[0013] 2. “Pore diameter” is herein abbreviated to “pd.”

[0014] 3. “Membrane thickness” is herein abbreviated to “mt.”

[0015] 4. “Radius of curvature” is herein abbreviated to “rc.”

[0016] 5. “High throughput screening” is herein abbreviated to “HTS.”

[0017] 6. “Cell-based high throughput screening” is herein abbreviatedto “CBHTS.”

[0018] 7. “Nanometer” is herein abbreviated to “nm.”

[0019] 8. “microliters” is herein abbreviated to “μl.”

[0020] 9. “micrograms” is herein abbreviated to “μg.”

[0021] 10. “Coefficient of variation” is herein abbreviated to “CV.”

[0022] 11. “Cell activity assay apparatus” is herein abbreviated to“CAAA”

[0023] 12. “tcc” herein abbreviates “total count of cells” introduced ata site.

[0024] 13. “U₁” herein abbreviates the quantity of light emitted fromthe upper volume of a site of a CAAA at the start of an assay. This isproportional to tcc.

[0025] 14. “L₁” herein abbreviates the quantity of light emitted fromthe lower volume of a site of a CAAA at the start of an assay, known asthe background.

[0026] 15. “U₂” herein abbreviates the quantity of light emitted fromthe upper volume of a site of a CAAA at the end of an assay.

[0027] 16. “L₂” herein abbreviates the quantity of light emitted fromthe lower volume of a site of a CAAA at the end of an assay.

[0028] 17. “CL₂” herein abbreviates the quantity of light emitted fromthe lower volume of a site of a CAAA, L₂, by subtracting the backgroundL₁.

[0029] 18. “cmc” herein abbreviates “completely migrated cells” which isproportional to CL₂.

[0030] 19. “cmc%” herein abbreviates percent of cmc with respect to thetotal cells introduced at that site, that is cmc/tcc.

[0031] 20. “pmc” herein abbreviates “partially migrated cells” which isproportional to U₁−(CL₂+U₂).

[0032] 21. “pmc%” herein abbreviates percent of cells that partiallymigrated with respect to the total cells introduced at that site, thatis pmc/tcc.

[0033] 22. “mc” herein abbreviates “migrated cells” which equalscmc+pmc.

[0034] 23. “mc%” herein abbreviates percent of mc, that is mc/tcc.

Definitions

[0035] 1. “chamber,” “well” or “volume,” as used herein with respect toCAAA means the three-dimensional area of the CAAA for holding fluidsamples.

[0036] 2. “off-axis,” as used herein with respect to pores in membranes,means pores that are incident to the membranes, when held flat, with anangle of incidence greater than 0° and less than 90°.

[0037] 3. “Strictly normal,” as used herein with respect to a beam of ERand a flat surface, means the entire beam of ER is perpendicular to thatsurface.

[0038] 4. “β-normal,” as used herein with respect to ER and a flatsurface, means a beam of ER some of which is strictly normal to thatsurface, and the remainder of which is incident within a range of angleswhere β is the largest such angle and β<90 degrees. In other words, aβ-normal beam of ER with respect to a surface is a cone of light havingβ as the largest angle of incidence as shown in FIG. 2.

[0039] 5. “Substantially normal,” as used herein with respect to ER anda flat surface, means that the ER is β-normal to said surface and β isless than 15°. (This is in recognition that most detection andquantification systems employed in cell activity assays have detectionbeams where β is less than 15°.)

[0040] 6. “Detection beam,” as used herein with respect to ER, means theER of an automated reader or detection and quantification systemdirected at the sites of the apparatus within a specified cone, from aspecified distance and from a specified aperture.

[0041] 7. “β-normal detection beam,” as used herein, means a detectionbeam using β-normal ER.

[0042] 8. “Ideal opaque,” as used herein with respect to a film, meansfilm that stops or blocks all ER.

[0043] 9. “R-opaque,” as used herein with respect to film or membrane,means a film or membrane that stops ER in the wavelength range R, whereR is specified by a pair of numbers in brackets which delimits a rangeof ER wavelengths expressed in nanometers.

[0044] 10. “R-opaque @P%,” as used herein with respect to film ormembrane, means film or membrane that stops greater than P% of ER inrange R, where P is a decimal number between 0 and 100 and representsthe percent of ER that is blocked. For example, one film used inembodiments of this invention blocks more than 99.0% of ER in thewavelength range between 400 and 580 nanometers. The same film blocks99.9% in the ER ranges between 480 and 490, and between 510 and 540nanometers. This film is herein referred to as “[400-580]-opaque @99.0%”and “[480-490]-opaque @99.9%” and “[510-540]-opaque @99.9%.”

[0045] 11. “Ideal-opaque,” as used herein with respect to membranes,means membranes such that (a) the film from which such membranes aremade is ideal opaque, (b) the pores of such membranes are straight andparallel, and (c) said pores are positioned or angled such that when themembranes are flat, strictly normal ER cannot pass straight through thepores.

[0046] 12. “β-normal-opaque,” as used herein with respect to amembranes, means membranes such that no a-normal ER can pass straightthrough any pore.

[0047] 13. “Substantially opaque,” as used herein with respect tomembranes, means membranes such that no substantially normal ER can passstraight through their pores.

[0048] 14. “Geometrically R-opaque @P%,” as used herein with respect tomembranes, means membranes that are (a) made of film that is R-opaque@P%, and (b) no strictly normal ER passes straight through any pore ofthe membrane. As shown in FIG. 1, the least angle of incidence α of theoff-axis pores of such a membrane must satisfy the following equation:

α>φ where φ=sin⁻¹ (pd/mt).  (1)

[0049] 15. “β-normal R-opaque @P%,” as used herein with respect tomembranes, means (a) the film of the membrane is R-opaque @P%, and (b),no β-normal ER passes straight through any pore. As shown in FIG. 2, theleast angle of incidence α of the off-axis pores of such a membrane mustsatisfy the following equation:

α>(β+θ), where θ=sin⁻¹ ([pd×cos β]/mt).  (2)

[0050] 16. “Substantially R-opaque @P%,” as used herein with respect tomembranes, means membranes that are (a) made of R-opaque film @P%, and(b) allow no substantially normal ER to pass straight through any pore.Thus the least angle of incidence α of the off-axis pores of such amembrane must satisfy equation (2), above, for β<15°.

[0051] 17. “Substantially perpendicular,” as used herein with respect topores in membranes, means pores that are incident to the membranes, whenheld flat, with an angle of incidence less than 15°.

[0052] Note that membranes that are R-opaque @P% are not necessarilysubstantially R-opaque @P%, β-normal R-opaque @P%, or geometricallyR-opaque @P%, since they may have perpendicular pores which allowsubstantially normal light to pass straight through them. Note that amembrane suitable for cell activity assays will preferably have Pgreater than 99.0%, and the pore diameter will be greater than 3 micronsso that the open area of the membrane formed by the pores will be largerthan 2% of the membrane. More specifically, membranes for theseapplications cannot be R-opaque at 99% if the pores are substantiallyperpendicular because ER substantially perpendicular to the surface willpass straight through the pores. If, on the other hand, the pores aresufficiently off-axis, and all other aspects remain the same, themembrane can be substantially R-opaque @P%, β-normal R-opaque @P%, andgeometrically R-opaque @P%

SUMMARY OF THE INVENTION

[0053] The present invention provides CAAA employing membranes andmethods for using the CAAA for HTS, CBHTS, and cell based screening, aswell as basic research in cell activity. In particular the presentinvention is a CAAA using a class of membranes that are substantiallyR-opaque @P%. The present invention also includes the membranes used inthe CAAA and methods for their fabrication.

[0054] Because the membranes of this invention are substantiallyR-opaque @P% to the ER wavelengths of the instruments used for detectionand quantification with CAAA, detection and counting of cells from oneside of the membrane will not be influenced by cells on the oppositeside of the membrane or by simultaneous or by subsequent detection andcounting of cells on the other side of the membrane. This yields moreaccurate results than can be obtained with prior art methods. Inparticular, this allows the use of a method of detection andquantification that eliminates the errors due to both the volumetricinaccuracies of pipetting and the variations in the distribution ofcells in the media in which the cells are suspended. This lowers the CVof the assays such that they are appropriate for HTS and CBHTS in drugdiscovery and development.

[0055] Tchao's membrane is made of film opaque to the wavelengths ofexcitation and emission of some fluorescent dyes. That is, the Tchaomembrane is R-opaque @P%, where R is the range of wavelengths used bythe detection and quantification system, and P is the percent of lightblocked. The Tchao membrane, however, is specifically required to havesubstantially perpendicular transverse pores. It is therefore neither a“geometrically R-opaque @P%” membrane, nor a “β-normal R-opaque @P%”membrane, where “β” is the largest angle of incidence of light in thedetection beam, and “R” is a range of wavelengths of light. It istherefore not a “substantially R-opaque @P%” membrane. ER from detectionbeams of standard detectors will pass straight through the pores sincethey are substantially perpendicular. Therefore, with membranes commonlyused for cell activity assays which have between 5% and 15% open area(the total area of the pores), the amount of light passing through thepores of the membrane is significant. The transmission of wavelengthsnormal to the surface of the membrane from the excitation beam will passthrough substantially perpendicular pores, and cells on the oppositeside of the membrane from the detection beam will be counted if they areover a pore. In other words, light that is β-normal where β is less than15° will cross the membrane, excite cells that are over pores which willemit light and be counted (since that light will pass back through thesubstantially perpendicular pores). Thus cells that have not migratedthrough the membrane will be stimulated to emit ER and will be counted,reducing the accuracy of the results. For kinetic assays, this is not aproblem for two reasons. First, the membrane's open area is only between5% and 15%, and the number of cells that are used for an assay can beset so that they cover only 10% of the membrane. This reduces the numberof a cells starting out over pores. Secondly, in a kinetic study, theimportant parameter is the rate of change, and the fact that cells onthe origination side of the membrane are counted when they are overpores means only that the detector will count them much earlier than itwould with an opaque membrane made out of the same film with pores thatare off axis so that the excitation beam cannot pass directly throughthem. On the other hand, for assays that measure more than just kineticsor do not measure kinetics, and where the method involves counting allthe cells on both sides of the membrane at different points in theassay, as in the methods described below, having substantiallyperpendicular pores will decrease the accuracy of the assay, andincrease the CV significantly.

BRIEF DESCRIPTION OF THE DRAWINGS

[0056]FIG. 1 is a schematic cross-sectional representation of a portionof one of the membranes of the present invention with strictly normalER.

[0057]FIG. 2 is a schematic cross-sectional representation of a portionof the membrane of the present invention with β-normal ER.

[0058]FIG. 3 is a schematic cross-sectional representation of a portionof the membrane of the present invention with curved pores and β-normalER.

[0059]FIG. 4a is a schematic representation of a process for making themembranes of the present invention with a web of film and an excimerlaser.

[0060]FIG. 4b is a schematic representation of a top view of afabrication process for making the membranes of the present inventionwith framed film and an excimer laser.

[0061]FIG. 4c is a schematic representation of a side view of thefabrication process of FIG. 4b for making the membranes of the presentinvention with framed film and an excimer laser.

[0062]FIG. 4d is a schematic representation of a process for makingcurved pore membranes of the present invention with an excimer laser.

[0063]FIG. 5a is a side view schematic representation of a secondprocess for making the membrane of the present invention using cyclotronbombardment (and subsequent etching.)

[0064]FIG. 5b is a top view schematic representation of the secondprocess for making the membrane of the present invention using cyclotronbombardment.

[0065]FIG. 6a is a schematic representation of a CAAA comprising apreferred embodiment of the present invention viewed from above.

[0066]FIG. 6b is an enlarged schematic cross-sectional view of a portionof the preferred embodiment of FIG. 6a.

[0067]FIG. 6c is a further enlarged schematic cross-sectional view of aportion of the preferred embodiment of FIG. 6a showing the pores of themembrane.

[0068]FIGS. 7a, and 7 a ₂ are a top view and a side view, respectively,of another preferred embodiment of the present invention.

[0069]FIG. 7b is an enlarged schematic cross-sectional view of a part ofthe preferred embodiment of the present invention of FIGS. 7a ₁ and 7 a₂.

DETAILED DESCRIPTION OF THE INVENTION Membrane Geometry

[0070]FIG. 1 is an enlarged cross-sectional view of a portion of anidealized embodiment of a membrane of the present invention drawn withstrictly normal ER 150. The membrane 100 of FIG. 1 has a first surface110, a second surface 120, and a plurality of pores 130. The pores ofmembrane 100 are off-axis such that the least angle of incidence of anypore is ox. The pore 130 has an angle of incidence such that thedetection beam cannot travel directly through the pore from the secondsurface to the first surface of the membrane or vice versa. As shown inFIG. 1, the least angle incidence, α, must be greater than φ, where φ isdefined by the equation:

φ=sin⁻¹ (pd/mt)  (1)

[0071] and where pd is the pore diameter and mt is the membranethickness. In this case, no ER emitted from a strictly normal detectionbeam can pass straight through the pores of the membrane.

[0072]FIG. 2 is an enlarged cross-sectional schematic view of a portionof the membrane of the present invention. The β-normal opaque membrane200 has a top surface 210, a bottom surface 220 and a plurality of pores230. Ray 260 emitted from a β-normal detection beam, having the maximumangle of incidence of the detection beam, β, is drawn in FIG. 2. Thepore diameter pd and the membrane thickness mt are as indicated. Theangle of a pore of the membrane of least angle of incidence is α.Membrane 200 is β-normal opaque, if and only if:

α>(β+θ), where θ=sin⁻¹ ([pd×cos β]/mt).  (2)

[0073]FIG. 3 is an enlarged cross-sectional schematic view of a portionof an embodiment of the membrane of the present invention having curvedpores 300. The top surface 310, the bottom surface 320, and a pore 330are indicated. Ray 360 emitted from a β-normal detection beam, havingthe maximum angle of incidence of the detection beam, β, is drawn inFIG. 3. The pore diameter pd and the membrane thickness mt are asindicated. The least angle of incidence, α, of a pore of the membrane isdetermined as indicated in FIG. 3. Membrane 300 is β-normal opaque, ifand only if Equation (2) is satisfied:

α>(β+θ), where θ=sin−1 ([pd×cos β]/mt).  (2)

Membrane Fabrication

[0074]FIG. 4a is a reduced cross-sectional schematic view of anapparatus used in fabrication of embodiments of the membrane of thepresent invention. The film from which the membrane is manufactured isin the form of a web on a roll called the unwind roll 410, which isunwound and held with a flat section 420 between the unwind roll 410 anda second roll, the rewind roll 430, which contains the fabricatedmembrane. The flat section of the film 420, between the unwind roll 410and the rewind roll 430, passes under multiple beams of excimer laserlight 440, each beam of which has a diameter slightly smaller than thepores that are being fabricated. How much smaller is determined by thethickness of the film, what the film is made of, and the wavelength ofthe ER from the laser. Excimer lasers are used because the wavelength oftheir ER is very short, typically between 200 nm and 400 nm. This allowspores of less than a micron to be made. However, the pore sizes ofpreferred embodiments of the present invention are 1.0, 2.0, 3.0, 5.0,8.0, 10.0, 12.0, and 14.0 microns, which are relatively easy to makecompared to the submicron pores. The beams of laser light 440 strike thefilm at angle α, as shown in FIG. 4a.

[0075] In preferred embodiments, α is between 15° and 70° depending onthe particular β-normal opaque membrane required, the membranethickness, pore diameter and the nature of the cell activity assays inwhich it is to be employed. In some assays, the shortest possible poresare the most desirable, and in others, longer pores are optimal. Forexample, if the assay is designed for a minimum incubation period, thepores are preferably short. On the other hand, if maximum sensitivity isparamount, longer pores may be better, since a lower number of negativecontrol cells will pass through the membrane.

[0076] The multiple beams of excimer laser ER are created by projectingthe output of the laser onto a mask which forms multiple discrete beams,e.g., thousands of beams, which are then focused on the film. The laserER ablates the film creating straight pores through the material. Thepower of the laser, the composition of the film material, and itsthickness, determines the duration of the ablation. The details are wellknown to those practiced in the art. One advantage of membranefabrication with a laser is that the exact position and number of thepores can be controlled. This is advantageous since it lowers the CV ofthe assays by removing the variation associated with different sites ofthe cell activity apparatus having different numbers and positions ofpores. It also may prove essential as cell-based assays are developedwhich require smaller and smaller volumes and areas for each site. Onepreferred embodiment of the present invention employs a microplate sizedapparatus (5.030″×3.365″ footprint) with 1536 sites of 1 mm diameter.Preferred embodiments of the β-normal opaque membrane employed in thisapparatus have pore diameters of 1, 2, 3, 5, 8, 10, 12, or 14 microns,and the thickness ranges between 5 and 50 microns. The pore densitiesrange from 1×10³ to 2×10⁷ pores/cm². The smaller the area of membraneper assay site, the larger the errors become that are associated withpore distribution irregularity. This negatively affects the CV of thewhole assay. Consequently, excimer laser fabrication with virtually novariation in the size, number, angle, and position of the pores, is animportant advantage.

[0077]FIG. 4b is a schematic oblique top view and FIG. 4c is a side viewof a preferred fabrication method of the membranes of the presentinvention. With this method, the membranes are constructed from film byablating pores with a laser apparatus as in FIG. 4a. In this methodframes of a CAAA 460 with the film 461 bonded to one side are preciselypositioned in an angled jig 470 with a sliding member 471 as shown inFIG. 4c. Multiple laser beams 440, each with a diameter proportional tothe diameter of the pores being fabricated, strike the film at an angleof incidence α, as indicated. As described above, Equation (2) must besatisfied to construct a β-normal opaque membrane. That is,

α>(β+θ), where θ=sin⁻¹ ([pd×cos β]/mt),  (2)

[0078] where mt is the film thickness, and pd is the pore diameter asshown in FIG. 2. If β<15 degrees the membrane thus manufactured will besubstantially opaque. The framed film 461 is moved in the directionindicated by arrow 475 in incremental steps under the laser beams 440,preserving the angle cc at which the laser beams strike the film. Thebeams 440 sequentially ablate rows of pores in a pattern defined by amask (not shown) that forms clusters of discrete beams in the opticalpath between the source of the laser light (not shown) and a focusinglens system 450 that focuses the clusters of laser beams on the film. Inthis embodiment of the laser fabrication apparatus, the laser beams 440are grouped into a pattern 480 corresponding to the sites of the CAAArepresented in FIG. 4b by the 32×48 array of 1536 sites. Thisfabrication technique has several advantages: (a) the number of pores ateach site is fixed and (b) said pores can be positioned in any fixed anduniform pattern across the whole framed film 461. This means that siteto site variation in CAAA using the framed membrane is practically zerowith respect to the membranes. It also means that the application of thehydrophobic mask of some preferred embodiments (described below) is nolonger as positionally and dimensionally critical to the uniformity ofthe sites, and hence to the CV of the assays.

[0079]FIG. 4d shows the method of fabrication of the membranes of thepresent invention from film in which the pores are machined, burned, orablated with a laser as in FIG. 4a. The film from which the membranesare manufactured is in the form of a web on an unwind roll 410, which isunwound and passes around secondary roller 490 where it changesdirection 90 degrees and passes around the fabrication roll 495 of verysmall diameter. The diameter of fabrication roll 495 is proportional tothe radius of curvature (rc) of the pores fabricated with this method:the smaller the diameter of 495 the smaller the rc of the pores. Thelaser ER strikes the film at a range of angles on a very narrow part ofthe area of the film where it is bent around the fabrication roller 495.The laser ER ablates straight holes in the bent film, so when themembranes are flat, the pores are bent (see FIG. 3) The membrane webthen makes a quarter turn around roll 491 and hence to the rewind roll430 where it is rewound as fabricated membrane. The film moves in verysmall incremental steps in the direction indicated by arrow 475, thestep increment being determined by the width of the area where the laserablates the pores in the film. The laser beams 440 sequentially ablaterows of pores, the diameter and pattern of which are defined by a mask(not shown). The range of angles of the pores will be proportional tothe diameter of the fabrication roll 495 and the width of the area wherethe laser beams 440 strike the film.

[0080]FIG. 5a is a reduced cross-sectional schematic view of a secondkind of apparatus used in fabrication of embodiments of the membranes ofthe present invention. The film from which the membrane is manufacturedis in the form of a web on unwind roll 510, which is unwound and heldflat 520 between the intermediate rolls 530 and 540, and then moves tothe rewind roll 550, which contains the irradiated membrane. The sectionof the film held flat and taught 520 between the intermediate rolls 530and 540 passes under a beam 560 of high energy charged particles emittedby a cyclotron (not shown). The diameter of the high energy particlebeam in one preferred embodiment is approximately 6 centimeters, and itis swept back and forth across the web by deflecting it with anelectromagnetic field from sweeping magnet 570 as shown in FIG. 5b. Thebeam sweeps back and forth across the width of the film in a pathperpendicular to the line of motion of the web. The beam (or fog ofions) is about 5 meters from sweeping magnet 570 when it penetrates thefilm. As the beam sweeps back and forth over the width of the film 520,the film moves under it at a steady rate which is determined by the poredensity desired for the membrane. As the film passes under the beam, thecharged particles pass through it, breaking the molecular bonds of thepolymer chains composing the film. The energy of the particles in thebeam ranges between 1 and 2 million electron volts and must besufficient to pass completely through the film at the angle of incidenceα of the beam. This angle α is set between 15° and 70°. The optimalangle for a particular β-normal opaque membrane is determined by themembrane thickness, the pore diameter, and the nature of the cellactivity assays in which it is to be employed.

[0081] In this fabrication technique, the second step of the process isnot shown and consists of submersing the irradiated film in an etchingbath. This etches pores along the straight paths of broken polymersthrough the film created by the high energy particles from thecyclotron. The diameter of the pores is determined by the duration ofthe etching process, and the temperature and concentration of theetching bath. The specifications for this process are well known tothose practiced in the art of fabricating track-etch membranes.

Cell Activity Assay Apparatus

[0082]FIGS. 6a-6 c are schematic diagrams of an embodiment of thepresent invention. The multi-site CAAA 600 has a β-normal R-opaque @P%membrane, specifically the substantially R-opaque @P% membrane describedabove. FIG. 6a is a top and side cross-section view of the wholeinstrument, and FIGS. 6b and 6 c are enlarged cross-section views ofportions of the apparatus.

[0083] The assay apparatus 600 comprises three rigid parts: an upperpart consisting of a transparent film 621 bonded to a rigid frame 620, amiddle part consisting of the membrane 651 bonded to a second rigidframe 650, and a bottom part consisting of a transparentinjection-molded microplate 680.

[0084] The membrane 651 is fabricated from film, which can be made froma variety of materials including plastic, metal, glass, ceramic, organicmaterial, or combinations thereof. The membrane pores 695 (illustratedonly in FIG. 6c) can be fabricated in a number of ways, two of which areillustrated and described above. The frame 650 can be plastic, steel,stainless steel, aluminum, or another suitable material. The frame mustbe rigid enough to keep the membrane, and any grids, coatings, orsite-delimiting devices attached thereto, substantially flat. Themembrane can be attached to the frame by any suitable fastening means,including glue, heat seals, ultrasonic seals, or mechanical means.

[0085]FIG. 6a shows the ninety six (96) assay sites 630 of CAAA 600arranged in an 8×12 array. Each assay site 630 comprises a discrete,delimited area 656 of membrane 651, along with two three-dimensionalcompartments (wells)—the upper volume or well 693, above the membrane651, and the lower volume or well 692, in the microplate 680 below themembrane 651.

[0086] The film 621 delimits the top of each upper volume 693 andcreates optically advantageous flat surfaces, which minimize reflectionand refraction of ER from detection beams and ER emitted from the upperwell of the test sites 630. An opaque mask 625, affixed to the bottomsurface of film 621, surrounds and separates the transparent spots 627at the tops of the assay sites 630. This mask is also hydrophobic andcircumscribes the top perimeters of the upper volumes 693.

[0087]FIG. 6c shows a greatly enlarged view of a section of the membrane651, which, with the frame 650, forms the middle part of the CAAA 600.FIG. 6c illustrates the off-axis pores 695 of substantially R-opaque @P%membrane 651. This figure also shows a membrane section at the edge ofan assay site 630 and illustrates the two hydrophobic masks that areaffixed to the membrane 651, one (655) on the top surface 653 and one(654) on the bottom surface 652. Hydrophobic mask 655 delimits thebottom perimeter of the upper volume of site 630, and circumscribes themembrane area 656 where cell activity can occur across the membrane.Hydrophobic mask 654 helps circumscribe the top surface of the lowervolume, as described below.

[0088] During an assay, the upper volume (upper well) 693 and lowervolume (lower well) 692 of each site 630 are filled with fluid solutionscontaining chemical compounds and/or biological cells in suspension asshown by the shaded areas in FIG. 6b and the cross-section view of

[0089]FIG. 6a. Surface tension of the fluid in each upper volume 693,along with the pair of hydrophobic masks 625 and 655, confineupper-volume fluid and create air space 697 surrounding and isolatingthe upper volumes. Hydrophobic coating 686, affixed to the rim 685 ofthe lower volume 692, forms a shield seal with the hydrophobic mask 654on the under side 652 of membrane 651. The membrane 651 is positionedand held on top of and against the rims 685 of the lower well 692, whichconfines the lower-volume fluid. The flat transparent bottoms 682 of thewells in the microplate 680 are optically advantageous surfaces throughwhich light can pass into and out of the lower volumes 692.

[0090]FIGS. 7a ₁ and 7 a ₂ show the top view and a side view,respectively, of a CAAA 700 having one thousand five hundred and thirtysix (1536) assay sites. In this embodiment the assay sites 730 arearranged in a 32×48 array within the footprint of a standard microplate(5.030″×3.365″). The apparatus 700 is composed of three rigid parts: anupper rigid frame 720, a middle rigid frame 750 and a lower rigid frame780. The upper rigid frame 720 is bonded to a transparent film 721. Themiddle rigid frame 750 is bonded to substantially R-opaque @P% membrane751. The lower rigid frame 780 is bonded to transparent film 781. Eachof the assay sites 730 has an upper volume 793 and a lower volume 792,as shown in FIG. 7b.

[0091]FIG. 7b is a cross-sectional, enlarged, schematic view of aportion of CAAA 700. The upper film 721 has a hydrophobic mask 725bonded to its bottom surface 723. The membrane 751 has a top hydrophobicmask 755 bonded to its top surface 753, and a bottom hydrophobic mask754 bonded to its bottom surface 752. The lower film 781 has a topsurface 782 with a hydrophobic mask 784 bonded to it.

[0092] Each assay site 730 consists of the following elements: thetransparent area 727 of upper film 721 and hydrophobic mask 725 on lowersurface 723 surrounding transparent area 727, the open area 756 ofmembrane 751 and hydrophobic mask 755 on the top surface 753 of membrane751 surrounding 756, and hydrophobic mask 754 on bottom surface 752 ofthe membrane 751, the transparent area 786 of the lower film 781, thehydrophobic mask 784 on the top surface 782 of the lower film 781, andthe upper volume 793 and the lower volume 792. The upper air space 797and lower air space 798 surround and separate each of the sites. In thisembodiment, the volumes 793 and 792 are between 0.5 μl and 2.5 μl, andthe distances between the centers of sites 730 is 2.25 mm.

[0093] Hydrophobic masks 725 and 784 are opaque and surround transparentareas of the top film 727 and bottom film 786, respectively, that aredirectly above and below each assay site. ER from the detection beams760 and 761 and the ER 762 and 763 emitted by the fluorescent dye in theupper and lower volumes pass in and out through upper transparent area727 and lower transparent area 786, respectively. Thedetector/quantification apparatus above the upper film 721 is composedof a housing 740, a fiber optic bundle 742 for collecting emitted ER 762from the top volume 793 and top surface 753 of the site 730, and anotheroptical conduit 744 for delivering the excitation ER 760 to the uppervolume 793 and upper surface 753 of the membrane of the sites 730. Thedetector/quantification apparatus below the lower film 781 is composedof a housing 741, a fiber optic bundle 743 for collecting emitted ER 763from the bottom volume 792 of the site 730, and another optical conduit745 for delivering the excitation ER 761 to the lower volume 792 andlower surface 752 of the membrane of the site 730.

[0094] The interfaces between the three rigid frames 780, 750, and 720,can be either sealed to gas exchange or not as required by the assay. Inmost cell-based assays, airflow needs to be minimized to preventexcessive evaporation, without overly inhibiting diffusion of oxygen andcarbon dioxide. This can be accomplished in a number of ways including:positioning a thin layer of open cell foam between the frames, providingmultiple small channels between the frames, providing film material for721 and/or 781 with sufficient gas exchange rates, providing microscopicholes in 721 and/or 781 between the test sites, or providing enoughmicroscopic pores in 721 and/or 781 at the test sites for sufficient gasexchange.

[0095] The materials from which suitable films can be made for thefabrication of opaque membranes include plastics, organics, metals,glass, ceramics and combinations thereof. In preferred embodiments ofthe opaque membrane of the present invention, polyester film is usedwith various dyes that make it opaque at various wavelength ranges thatmatch fluorescent excitation and emission bands of the various dyes usedto tag or label the cells used in the cell activity assays. Theembodiments, described above, manufactured with the cyclotronbombardment and etching technology require materials that can be etched.Polyester and polycarbonate have excellent etching characteristics andare widely used. Polyester is preferred because it is easy to introducedye into the film after it is initially fabricated. Polycarbonate filmis now fabricated with dye incorporated into the basic film, but at thistime the other characteristics of this film, in particular theuniformity of its thickness, make it a poor candidate.

[0096] Coating or depositing metallic layers on plastic films and/ormembranes is another method of making the membranes opaque. There areadvantages and disadvantages to this fabrication technique. For example,with a dyed substantially opaque membrane, some light passes through thepores via reflections within the pores. The amount of light which passesthrough, however, is insignificant and is not a practical problem sincestandard detection/quantification systems used for cell activity assaysare not sensitive enough to measure it. If the membrane is coated withmetallic atoms, however, the interior surfaces of the pores can beextremely reflective. This is counter-productive to the goal offabricating a substantially R-opaque @P% membrane where P is above99.9%. Furthermore, the deposited coating must not interfere or affectthe activity of the cells used in the assays.

Methods of Using Cell Assay Apparatus

[0097] In one method for using the embodiment of the invention, shownschematically in FIGS. 6a-6 c above, cells to be interrogated arepositioned on one side of the membrane and solutions to be assayed fortheir influence on cell activity are positioned directly opposite on theother side of the membrane. The preferred membrane of this embodiment issubstantially R-opaque @P%, where R is 400 nm-580 nm and P% is 99.9% andthe membrane thickness is 31 microns, the pore diameter is 8 microns,the range of angles of incidence of the off axis pores is between 44°and 46°. When using such a CAAA with 485 nm ER for excitation and 530 nmdetection/quantification systems for ER emitted by the cells in thesites, the contribution of the cells on the opposite side of themembrane from the excitation/detection/quantification system is lessthan 0.0005% of the count at any site. The membrane could have a poredensity between 1×10³ and 1×10⁹ pores/cm², a thickness between 1 and1000 microns, and pores with angle of incidence between about 15° and70°.

[0098] Several methods using CAAA 600 or similar apparatus are describedin more detail below.

Method I

[0099] Method I comprises the following steps:

[0100] 1. About eighty-four of the lower volumes (wells) 692 are filledwith 30 μl of test solutions, i.e., positive controls, negativecontrols, or unknowns.

[0101] 2. The remainder—about one row of lower plate 680—are filled with25 μl of cell suspension in a serial dilution down to a cellconcentration at or just below the lower sensitivity limit of thedetection/quantification system. This provides a standard with which tocompensate for reader error when the lower volume of the test sites areread.

[0102] 3. The rigid framed membrane 650/651 is positioned and attachedto the microplate.

[0103] 4. About 25 μl of cell suspension is applied to the uppersurfaces 653 of membrane 651 in about eighty-four of the upper volumes693 of the test sites. 25 μl of the same cell suspension in a serialdilution is then added to the remaining upper sites as in step 2. Thisprovides a standard with which to compensate for reader error when theupper volume of the test sites are read.

[0104] 5. The lid 620/621 is positioned and attached to the CAAA.

[0105] 6. An automatic fluorescence reader is used to read assembledCAAA 600 and determine values for U₁ and L₁ at each site. The data arestored in memory for later comparison, compensation, and computation.

[0106] 7. CAAA 600 is incubated at the appropriate temperature andhumidity (in human CAA, 37° C. and 98% relative humidity is generallyappropriate), and the appropriate interval of time for the particularcell activity assay. This period is usually between 15 minutes and 48hours. The optimal incubation period is determined by doing kinetics ora sequence of experiments with different incubation times, and thenselecting the incubation period that gives the optimal result. In a cellactivity assay this usually means the result where the differencebetween counts at the negative control sites, and at the positivecontrol sites is maximized. In HTS, however, where the longer the timeof incubation, the more expensive the assay, the optimum incubationmight be much shorter. It might be the shortest time required todetermine whether a significant difference exists between negativecontrol sites, positive control sites and sites with the compounds beingscreened.

[0107] 8. CAAA 600 is removed from the incubator and read a second timeas described in step (6) and the values U₂ and L₂ for each site and thedata are stored in the computer memory.

[0108] 9. The computer calculates the cmc, cmc%, pmc, pmc%, mc, and mc%from the data collected in steps 6 and 8. The computer can alsocompensate for the errors in the detection/quantification system byusing the data collected from the row of sites with the serial dilutionsof the cell suspension. It then calculates the averages of theseabove-mentioned figures for each set of replicate sites (usually between2 and 4) and compiles the results of the whole assay comparing positivesite sets, negative site sets and unknown site sets. It also calculatesthe CV for the assay. The calculated results are then available forfurther analysis.

Method II

[0109] Method II comprises the same steps as Method I, in addition tothe following step, step 7 a, performed after step 7:

[0110]7 a. Kill all the cells in the upper wells, or kill all the cellsin the apparatus, by (1) irradiating them with short wavelength ER, e.g,254 mn, (2) removing the lid 620/621 and replacing it with another lidwith about 10 μl of a solution on each site on the bottom surface 623 ofthe film 621, that rapidly kills the cells, or (3) some equivalentmethod.

Method III

[0111] Method III comprises the same steps as Method I, in addition tothe following step, step 7 b, performed after step 7:

[0112]7 b. Immobilize all the cells in the upper wells or in the entireapparatus by; (1) freezing the cells (usually accomplished by freezingthe entire apparatus), (2) rapidly lowering the temperature of the cells(or apparatus) to between 0° C. and 4° C., (3) removing the lid 620/621and replacing it with another lid with solutions affixed to all thesites on the lower surface of film 621 that immobilize the cells, e.g.,4 millimolar EDTA, or (4) some equivalent method.

[0113] Methods II and III are preferred over Method I in cases where thecell activity is so rapid that the accuracy of the results will becompromised by the difference in elapsed time the cells are active atdifferent test sites. This can be very significant if the incubationtime is short, e.g., less than thirty minutes, and thedetection/quantification time is long, e.g., ten minutes. With the largenumber of sites used for HTS and ultra-HTS (e.g., 1536 and 3456 sites)the cells must be killed or immobilized for accurate results.

Methods IV through VI

[0114] Method IV is a simplified version of Method I. In this method,all of the steps of method I are performed, with the exception of step(6). Thus, in this simplified version, the readings of the emissionsfrom the upper and lower wells are obtained only once.

[0115] Method V is a simplified version of Method II and Method VI is asimplified version of Method III. That is, as described above, thereadings of step (6) are not performed.

[0116] These simplified Methods IV through VI will not yield nearly asmuch data, and the information extracted from the data will not be asrich or sensitive, but it may be sufficient in some screening contexts.The advantages of these simplified methods are that they are faster andless expensive than Methods I through III.

Method VII

[0117] Method VII is essentially a class of methods. Method VIIcomprises all of the Methods I through VI, using an apparatus having amembrane that is R-opaque @P%, but not substantially R-opaque @P%. Thatis, the membrane used in this class of methods has pores that allowsubstantially normal ER, such as that used by state-of-the-artdetection/quantification systems, to pass straight through the membrane.This will yield adequate results in some CBHTS contexts, although thesensitivity will be lower, particularly in comparison with Methods Ithrough III.

Method VIII

[0118] Method VIII is another class of methods. These methods use a CAAAsuch as that disclosed above and illustrated in FIGS. 7a ₁, 7 a ₂, 7 b,and 7 c. In these methods the first five steps of Method I through VIIare modified as follows:

[0119]1 a. The various control solutions, positive and negative, andtest solutions, e.g., chemotactic factors, are prepared cold. Thesolutions contain a sufficient proportion of collagen, gelatin,fibernectin, lamanin, or other appropriate gel-forming materialcompatible with the cells of the assay, to gel at temperatures of about20° C. to 40° C. The solutions do not gel when held between about 4° C.and 110° C., so they can be prepared in advance and stored at 4° C. Whenthey reach between about 20° C. and 37° C., they gel. Temperature, pH,and concentration of the gelling agents determine the amount of timerequired to gel. These parameters can be manipulated for conveniencewithin certain limits. For example, approximately 100 μg of Type Icollagen in one ml of DMEM buffer at pH 7.2 is a good medium for manycell activity assays. (See M.E. Steams et. al, Clinical Cancer Research,Vol. 5, March 1999). Identical volumes between 0.5 μl and 2.5 μl of coldsolutions containing gel-producing compounds mixed with the compounds tobe tested e.g., positive controls (known chemotactic factors), negativecontrols with no additional compounds, and unknowns, are pipetted ontothe top surface 782 of the bottom film 781 surrounded by hydrophobicmask 784 that defines the bottom of the lower volume of the test sites730 with a pipetting robot (e.g., Model C-300 or Model C-400, Cyberlab,Inc., Brookfield, Conn.). About 1500 sites of the available 1536 arefilled. The droplets of solution adhere to the film 781 and rise between1.0 mm and 1.7 mm above the top surface 782. The hydrophobic masksvisually define the locations of the cell activity test sites andprevent the lateral movement of the test solutions.

[0120]2 a. The remainder of the test sites are filled with a serialdilution of the cell suspension, but in this case the same volume isused as in step (1 a). Other kinds of control and calibration solutions,e.g., fluorescent dyes, can also be placed at various positions on theapparatus to facilitate detection of errors in the various components ofthe system and to enable compensation for such errors.

[0121]3 a. The rigid framed membrane 750/751 is then positioned over andattached to the lower framed film 780/781. The alignment and attachmenthardware or bonding agents fix the lower and middle components of theapparatus together. The final distance between the membrane and thebottom film is determined by the thickness of the portion of the framesbetween the top surface of the lower film 782 and the bottom surface ofthe membrane, 752 and is such that the solutions on the lower filmcontact and wet the lower surface 752 of the membrane 751. If volumes atthe low end of the range are desired, this distance is decreased, andvice versa.

[0122]3 b. These two components 780 and 750 now attached together areset aside or placed in an incubator for a period between 10 and 90minutes to allow the solutions to gel.

[0123]4 a. Between 0.5 μl and 2.5 μl of the cell suspension is thenpipetted onto about 1500 sites on the top surface 753 of the membrane751. In this case, however, the sites with the serial dilutions of thecell suspension and the other sites with control and calibrationsolutions have no cell suspension solutions applied. This is because inCAAA 700, the solutions at these sites in the lower wells make contactwith the bottom side 754 of the membrane, which is not the case withCAAA 600.

[0124]5 a. The lid 721/720 is then positioned over the framed membrane750/751 and placed down on the two lower components where it is attachedby the attachment hardware or bonding agent. The lower surface 723 ofthe upper film 721 makes contact with approximately 1500 volumes of cellsuspension, and the three rigid components of the apparatus 720, 750,and 780 become a unit 700.

[0125] At this point in the procedure, any of Methods I through VIIIcommencing with step (6)(as described above) can be followed, making thenecessary changes in the procedure required by the changes in thehardware. For example, the detector beams of the automatic fluorescentreader have to be smaller, as would the optical collection system thatcollects and measures the amount of light emitted from the upper andlower volumes of the sites.

[0126] The methods described above, using apparatus 600 or 700 orsimilar apparatus, allow the detection and quantification at each siteof (a) the number of cells pipetted, (b) the number of cells that havemigrated through the membrane, (c) the number that have not migrated,(d) the number that have migrated into the membrane but have not passedthrough, and (e) the total number that have migrated. From this data,the percent of the cells that have migrated at any site can becalculated as well as the percent of cells that have passed through themembrane at that site. Since these results are calculated independentlyfor each site, both the errors associated with pipetting (variations involume) and the errors associated with the uneven distribution of cellsin a unit volume are eliminated.

[0127] If the substantially R-opaque @P% membrane used in apparatus 600or 700, was fabricated according to the method disclosed in FIG. 4b andFIG. 4c, further reduction in the CV is achieved due to completeuniformity in the number of pores from site to site.

[0128] When Method VIII is used with CAAA that employ R-opaque @P%membrane that is not substantially R-opaque @P%, the results will beadequate in some CBHTS contexts. These include assays where it isdesirable to use (a) very low pore densities, (b) large pore diameters,(c) thin membranes, and (d) low cell densities. The system must alsohave very low background fluorescence and the detection/quantificationsystem must be very sensitive. In these contexts, the detection systemdetects cells when they migrate over pores. New detection instrumentsare being developed e.g., Cellomics (Pittsburgh, Pa.) which have highresolution optical systems that may accomplish this. Migration of cellsin this context would not be measured by how many cells passed throughthe membrane, but how many migrated over the top of a pore. Such assaysare rare and expensive. When this CBHTS context exists, the methods usedwould most likely be designed to acquire data about the kinetics of cellactivity, and these methods are different from the ones described here.

[0129] All of the above methods can be modified so that the cells areintroduced into the lower well of the CAAA. For example, in Method VIII,the cells can be suspended in an ungelled gel solution, as describedabove, and applied to the sites on the bottom side of the membrane whenit is inverted. The inverted lower framed film is then positioned overand attached to the inverted framed membrane. When the solutions gel,the apparatus is inverted and test solutions and controls are applied tothe top sites of the membrane, and then the upper framed film ispositioned and attached. The cells now migrate up through the filter.Similar results can be achieved with CAAA similar to 600, making thechanges necessary to accommodate the differences in the apparatus.

[0130] Many variations and modifications of the embodiments describedherein will be obvious to one of ordinary skill in the art in light ofthe above disclosure. For example, in an alternative CAAA, each site isa well provided with an insert which divides the well into an upperchamber and a lower chamber. In some such CAAA the inserts are attachedtogether in the form of a plate. The insert incorporates the membrane ofthe present invention.

[0131] As another example, in some cases, chemical interactions betweenthe cell sample and a chemical in the sample solution or chemicalintroduced into the solution after cell movement through the membranecreates light which is detected without introducing an ER beam. In suchcases, the pores would be angled to block transmission of ER that may beemitted by nonmigrated cells if the chemical diffuses into the chamberon the opposite side of the membrane. Further, the present inventioncould also encompass assays in which the sample is not necessarily in aliquid medium but, for example, may be carried by a gas medium so longas the membrane is substantially opaque to the ER wavelengths introducedand detected.

[0132] The foregoing disclosure of embodiments and methods of thepresent invention has been presented for purposes of illustration anddescription. It is not intended to be exhaustive of to limit theinvention to the precise forms disclosed.

The scope of the invention is to be defined only by the claims appendedhereto, and by their equivalents. What I claim is:
 1. A cell activityassay apparatus using electromagnetic radiation detection beams tomeasure cell activity, comprising at least two chambers separated by amembrane, wherein the membrane has a first surface, a second surface, athickness mt, and a plurality of off-axis pores having a diameter pd,wherein the off-axis pores traverse the film from the first surface tothe second surface at an angle α with respect to a line normal to thefirst surface of the film.
 2. The cell activity assay apparatus of claim1, wherein the angle α is between about 15 degrees and about 70 degrees.3. The cell activity assay apparatus of claim 1, wherein the angle α issuch that a detection beam of electromagnetic radiation, incidentstrictly normal to the first surface of the membrane, does not passbetween the first surface and the second surface.
 4. The cell activityassay apparatus of claim 1, wherein the angle α is greater than anangle, φ, wherein φ is defined by the equation, φ=sin⁻¹(pd/mt).
 5. Thecell activity assay apparatus of claim 1, wherein the angle α is suchthat a detection beam of electromagnetic radiation, having a range ofangles of incidence, and incident substantially normal to the firstsurface of the membrane, does not pass between the first surface and thesecond surface.
 6. The cell activity assay apparatus of claim 1, whereinthe angle α is greater than the sum of an angle β and an angle, θ,wherein β is the maximum angle of incidence of the detection beam withrespect to a line normal to the first surface, and θ is defined by theequation θ=sin⁻¹([pd=cos β ]/mt).
 7. The cell activity assay apparatusof claim 1, further comprising a rigid frame attached to the membrane,such that the membrane is held flat by the rigid frame.
 8. A membranefor use in cell activity assay apparatus using electromagnetic radiationdetection beams to measure cell activity, comprising a film having afirst and second surface, a thickness mt, and a plurality of off-axispores having a diameter pd, wherein the off-axis pores traverse the filmfrom the first surface to the second surface at an angle α with respectto a line normal to the first surface of the film.
 9. The membraneaccording to claim 8, wherein the angle α is about 3 degrees and about70 degrees.
 10. The membrane according to claim 8, wherein the angle αis such that a detection beam of electromagnetic radiation, incidentstrictly normal to the first surface of the film, does not pass betweenthe first surface and the second surface.
 11. The membrane according toclaim 8, wherein the angle α is greater than an angle, φ, wherein φ isdefined by the equation, φ=sin⁻³¹ ¹(pd/mt).
 12. the membrane accordingto claim 8, wherein the angle α is such that a detection beam ofelectromagnetic radiation, having a range of angles of incidence, andincident substantially normal to the first surface of the film, does notpass between the first surface and the second surface.
 13. The membraneaccording to claim 8, wherein the angle α is greater than the sum of anangle β and an angle θ, wherein β is the maximum angle of incidence ofthe detection beam with respect to a line normal to the first surface,and θ is defined by the equation θ=sin⁻¹([pd×cos β ]/mt).
 14. A methodfor measuring cell activity, comprising the setps of: (a) providing acell activity assay apparatus having a poruous opaque membrane, having afirst surface and a second surface, separating a first fluid samplecontaining a plurality of cells in contact with the first surface, froma second fluid sample, in contact with the second surface; (b)incubating the cell activity assay apparatus; (c) directing a first beamof electromagnetic radiation having a first wavelength toward the firstsurface of the membrane through the first fluid sample; (d) measuring afirst quantity of electromagnetic radiation having a second wavelengththereby emitted; (e) directing a second beam of electromagneticradiation having a first wavelength toward the second surface of themembrane through the second fluid sample; and (f) measuring a secondquantity of electromagnetic radiation having a second wavelength therebyemitted.
 15. The method of claim 14, further comprising a step of takingan initial reading, wherein the initial reading is completed prior tothe incubating step of claim 14 and wherein the initial reading stepcomprises the steps: (i) directing the first beam of electromagneticradiation toward the first surface of the membrane through the firstfluid sample; (ii) measuring an initial first quantity ofelectromagnetic radiation having a second wavlength thereby emitted;(iii) directing the second beam of electromagnetic radiation toward thesecond surface of the membrane through the second fluid sample; and (iv)measuring an initial second quantity of electromagnetic radiation havinga second wavelength thereby emitted.
 16. The method of claim 14, furthercomprising the step of killing the plurality of cells, wherein killingthe plurality of cells is initiated after the incubating step of claim14.
 17. The method of claim 16, wherein the plurality cells are killedby exposing the plurality of cells to a quantity of electromagneticradiation having a short wavelength.
 18. The method of claim 17, whereinthe quantity of electromagnetic radiation has a wavelength between about200 nanometers and about 300 nanometers.
 19. The method of claim 14,further comprising the step of immobilizing the plurality cells, whereinimmobilizing the plurality of cells is initiated after the incubatingstep of claim
 14. 20. The method of claim 19, wherein the pluralitycells are immobilized by reducing the temperature of the plurality ofcells.
 21. The method of claim 19, wherein the plurality cells areimmobilized by introducing a chemical into the first fluid sample. 22.The method of claim 21, wherein the chemical is EDTA.
 23. The method ofclaim 14, wherein the sample of cells are labeled with a dye.
 24. Themethod of claim 23, wherein the dye is selected to emit electromagneticradiation having the second wavelength when exposed to electromagneticradiation having the first wavelength.
 25. The method of claim 14,wherein the membrane has a thickness, mt, and the plurality of off-axispores have a diameter, pd, wherein the off-axis pores traverse themembrane from the first surface to the second surface at an angle α withrespect to a line normal to the first surface of the membrane whereinthe angle α is greater than the sum of the angle β and an angle, θ,wherein β is the maxim angle of incidence of the first beam with respectto a line normal to the first surface, and θ is defined by the equationθ=sin⁻¹([pd×cos β]/mt).
 26. The method of claim 14, wherein the firstwavelength comprises a first range of wavelengths; and the secondwavelength comprises a second range of wavelengths.
 27. A method formeasuring cell activity, comprising, the steps of: (a) providing a firstchamber for receiving a first fluid, wherein the first chamber has anupper rim; (b) depositing the first fluid sample into the first chamber;(c) coating the upper rim with a first hydrophobic compound; (d)providing a membrane having a lower surface, an upper surface, and aplurality of off-axis pores traversing the membrane from the uppersurface to the lower surface; (e) coating a first area of the lowersurface of the membrane with a second hydrophobic compound, such thatthe first area coated has the same size and shape as the upper rim ofthe first chamber; (f) covering the first chamber with the membrane suchthat the first hydrophobic compound is adjacent to the secondhydrophobic compound; (g) coating the upper surface of the membrane witha third hydrophobic compound surrounding a second area of the membranethereby creating a second chamber for receiving a second fluid samplehaving a plurality of cells, such that the second chamber is directlyopposite the first chamber; (h) depositing the second fluid samle intothe second chamber; (i) incubating the first and second chamberscontaining the first and second fluid samples; (j) directing toward thefirst chamber a first beam of electromagnetic radiation of a firstwavelength, having a range of angles of incidence such that the firstbeam is substantially normal to the lower surface of the membrane; and(k) measuring a first quantity of electromagnetic radiation having asecond wavelength thereby emitted.
 28. The method of claim 26, furthercomprising the step of covering the second chamber with a transparentfilm after the step of depositing the second fluid sample into thesecond chamber.
 29. The method of claim 27, wherein the transparent filmcovering the second chamber has a lower surface in contact with thesecond fluid sample.
 30. The method of claim 26, wherein the pluralityof cells are labeled with a dye.
 31. The method of claim 29, wherein thedye is selected to emit electromagnetic radiation having the secondwavelength when exposed electromagnetic radiation having the firstwavelength.
 32. The method of claim 26, wherein the membrane preventspassage of the detection beam between the first surface and the secondsurface.
 33. The method of claim 26, wherein the membrane has athickness, mt, and the plurality of off-axis pores have a diameter, pd,wherein the off-axis pores traverse the membrane from the first surfaceto the second surface at an angle α with respect to a line normal to thefirst surface of the membrane wherein the angle α is greater than thesum of an angle β and an angle, θ, wherein β is the maxim angle ofincidence of the first beam with respect to a line normal to the firstsurface, and θ is defined by the equation θ×sin⁻¹([pd×cos β]/mt). 34.The method of claim 26, further comprising a step of taking an initialreading, wherein the initial reading is completed prior to theincubating step of claim 26 and wherein the initial reading stepcomprises the steps: (a) directing toward the second chamber a secondbeam of electromagnetic radiation of the first wavelength, having arange of angles of incidence such that the second beam is substantiallynormal to the upper surface of the membrane; and (b) measuring aninitial first quantity of electromagnetic radiation having the secondwavelength thereby emitted.
 35. A method for fabricating membranes foruse in cell activity assay apparatus comprising: (a) directing toward afilm a laser beam emitted by an excimer laser and having a wavelengthbetween 200 nm and 400 nm at a mask and forming a multiple discretelaser beams; (b) focusing the multiple discrete laser beams on a filmmaterial at an angle α with respect to a line normal to the film,wherein the angle α is between 15° and 70°; and (c) creating poresthrough the film by ablation.
 36. The method of claim 34, wherein thepore diameters are between 1 and 14 microns.
 37. The method of claim 34,wherein the density of pores in the membranes ranges from 1×10³ to 2×10⁷pores/cm².
 38. The methods of claim 34, wherein the membrane is opaquefor a predetermined range of wavelengths.
 39. A method for fabricatingmembranes having straight off-axis pores for use in cell activity assayapparatus comprising exposing a polymer film to a stream of high energycharged particles, wherein the stream of high energy charged particlesstrikes the film at an angle α with respect to a line normal to thefilm, thereby creating a track of broken polymers; and etching thetracks of broken polymers, thereby creating straight off-axis pores inthe membrane, wherein the angle α is between about 15° and 70°.
 40. Themethod of claim 38, wherein the membrane is opaque for a predeterminedrange of wavelengths.